Mechanical pretreatment of lignocellulosic biomass toward enzymatic/fermentative valorization

Summary Lignocellulosic biomass (LCB) has the potential to replace fossil fuels, thanks to the concept of biorefinery. This material is formed mainly by cellulose, lignin, and hemicellulose. To maximize the valorization potential of this material, LCB needs to be pretreated. Milling is always performed before any other treatments. It does not produce chemical change and improves the efficiency of the upcoming processes. Additionally, it makes LCB easier to handle and increases bulk density and transfer phenomena of the next pretreatment step. However, this treatment is energy consuming, so it needs to be optimized. Several mills can be used, and the equipment selection depends on the characteristics of the material, the final size required, and the operational regime: continuous or batch. Among them, ball, knife, and hammer mills are the most used at the laboratory scale, especially before enzymatic or fermentative treatments. The continuous operational regime (knife and hammer mill) allows us to work with high volumes of raw material and can continuously reduce particle size, unlike the batch operating regime (ball mill). This review recollects the information about the application of these machines, the effect on particle size, and subsequent treatments. On the one hand, ball milling reduced particle size the most; on the other hand, hammer and knife milling consumed less energy. Furthermore, the latter reached a small final particle size (units of millimeters) suitable for valorization.


INTRODUCTION
The growth of the global population, which is expected to reach 8.5 billion in 2030 (De Bhowmick et al., 2018), has led to an increase in energy use, and fossil fuels are one of the primary sources of energy. Thus, its usage has increased as well. Energy consumption is expected to rise a 50% from 2020 to 2050 (Office of Energy Analysis, 2019). Therefore, in recent years, environmental concerns and the scarcity of fossil fuels have resulted in the search for alternative energy sources (Danso et al., 2022;Silva et al., 2022). Biofuel is one of the alternatives for the replacement of fossil fuels. In fact, according to the International Energy Agency (IEA), the global demand for biofuels is expected to increase a 28% from 2021 to 2026 (IEA, 2021). LCB is the most used raw material for biofuel production. Therefore, it has become of interest as an environmentally friendlier way to obtain energy, chemicals, and bioplastics, when compared to fossil fuels, and has the potential to replace the exploitation of this kind of resource (Khoo et al., 2020), having a positive impact on the environment (Gundupalli et al., 2022). LCB comprises every plant and tree, either from forestry, agriculture, or as residue, and it is formed mainly by cellulose, hemicellulose, and lignin. Furthermore, it is renewable, biodegradable, and available (Guiao et al., 2022). Additionally, LCB can have zero net CO 2 emissions (Bastidas et al., 2022;de Freitas et al., 2021) as trees and plants act as CO 2 sinks (Saifuddin et al., 2020).
Cellulose is a homopolymer formed by units of glucose linked together by a b 1,4-glycosidic bond, and it is the most abundant natural polymer on earth (Arce et al., 2020). Hemicellulose is a heteropolymer formed mainly by five-carbon sugars (Joy and Krishnan, 2022). Sugars can be obtained from both compounds and can be further used to produce biofuels, bioplastics, and other value-added chemicals. Lignin is another biopolymer, but it is formed by aromatic compounds, more specifically: p-hydrofenyl, guaiacyl, and syringyl units (Dou et al., 2021), and it is a source of antioxidants (Xiao et al., 2021). Figure 1 shows the precursors of lignin and cellulose monomer.
Pretreated biomass can be valorized through several paths, depending on the final use: Chemically, enzymatically, fermentative, and thermally. The chemical path would aim to obtain chemical compounds mainly from the degradation of sugars contained in the cellulose matrix (Ji et al., 2018b); enzymatic and fermentative treatments can be performed separately (Ezeilo et al., 2017); however, they are usually performed together (Chen et al., 2015(Chen et al., , 2016. Initially, enzymatic treatments aim to concentrate sugars so the fermentation process can increase its yield as glucose would be the substrate for the microorganisms to grow (Vasic et al., 2021). Finally, the objective of thermal treatments is generally to either obtain biochar (Rozenfelde et al., 2017) or products derived from pyrolysis (Liang et al., 2021).
This work recollects information on several types of equipment used to perform mechanical size reduction at a laboratory scale. It also shows the effect on particle size and the improvement of the following treatment. This information helps decide which kind of mill to use depending on the material, particle size required, and final valorization option. Additionally, it includes the authors' critical review with considerations regarding scale-up from lab scale to industrial scale. In this section, the mechanical pretreatments performed on LCB are explained. Mechanical treatments are classified depending on the size reduction mechanism, generally applied to the biomass by an external body. When considering lignocellulosic biomass (LCB), cutting, shearing, compression, tearing, and breaking are the main mechanisms. Figure 2 shows a schematic description of the different mechanisms.
Cutting: This mechanism occurs when the comminution machine has a sharp end.
Shearing occurs between flat surfaces: one is fixed and the other moving. Usually, a gap is left between the two parts, so the comminuted material cannot pass through until adequate particle size is obtained.
Compression crushes de material with continuous vertical force. This mechanism is more suitable to be used for brittle material.
Tearing occurs when the moving part slides horizontally on the LCB and against the non-moving part.
Breaking uses dynamic compression force to comminute biomass. Figure 3 shows the different mechanical pretreatments found in the bibliography in recent years, from 2019 to 2022, mainly from the Scopus database. This search was initially performed using the following keywords: Mechanical treatments, Particle size reduction, Mechanical pretreatment biorefinery, and Ball milling. These keywords gave the result of more than 130 articles. After eliminating those articles that did not fall into the topic, the number of articles was 67 (without considering review articles). Some references from further years are also included as they were interesting.
The classification from Figure 4 is further explained in the next part of the article. However, this illustration helps to understand the differences between the mills.
Regarding the feedstock used by researchers in the bibliography consulted, LCB that comes from plants is the most used, specifically from agricultural and industrial wastes. Using this kind of feedstock shows the effort that researchers are making to valorize these residues and, as a result, become environmentally friendlier. Figure 5 shows the different biomass used as raw material.
Feedstock from plants is the most used for mechanical treatments. Residues from corn (24%) and wheat (21%) are widely used and account for 45% of all the residues used. Others from plants include grass, seeds, alfalfa cotton, and other residues. Sugarcane and rice residues accounted for 4% and 6%, respectively. As stated previously, LCB's main compounds are lignin, hemicellulose, and cellulose. These compounds can be valorized into various products, from biofuels (through fermentation) to biochar (through thermal treatments). Additionally, the availability of this kind of material makes it an excellent option to be valorized.

EQUIPMENT USED FOR CONTINUOUS MILLING
Mills that work under this configuration allow the material to go through the mill continually through the size reduction chamber in endless mode. It allows us to perform the size reduction with more quantity of raw material and, as a result, saves time and energy. Mills that work under this regime are explained next.

Disc refiner
The equipment used for disc refining consists of two dented discs. One disc is static, thus not moving, and the other is connected to a rotor. Both discs are equipped with dented or specially shaped active size reduction tools. The main variables to control the final particle size are the disc's rotational speed and the gap between the discs. Skinner et al. (2020) used this technology (aided with pressure) for the pretreatment and subsequent enzymatic hydrolysis of wheat straw. Biomass is fed into the center of the discs. Acceleration force moves biomass to the disc perimeter. Biomass radially flowing in disc gap is mechanically reduced in size between active working tools by shearing and tearing. The thinner the opening, the more intensive is the biomass size reduction.
Nevertheless, the high energy dissipation rate is usually recognized that can thermally degrade biomass components. Moreover, the disc gap also often tends to be plugged in case of fibrous and wet materials milling. It is, therefore, suitable to comminute dry biomass. Figure 6 shows an example of a disc refiner (Kratky, 2020).

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Furthermore, when the acidogenesis liquid phase was added to the biomass before size reduction, biomethane yield increased by 43% (Ma et al., 2021). This method can also be applied to produce biochemicals by pyrolysis treatment. It was demonstrated that after disc refining, pyrolysis led to a higher quantity of sugars and a metal ion reduction when compared to the raw material, leading to an improved pyrolysis product with no agglomeration of char (Torr et al., 2020). From the bibliography consulted, particle size after this operation is usually around 700 mm . However, if a smaller size is needed, other authors have reached particle sizes of around 120 mm (De Assis et al., 2018;Kim et al., 2016). Table 1 shows the references consulted on disc refiner as comminution process.

Screw extruder
Screw extrusion is an attractive technology for wet biomass processing, unlike disc refiner. Wet biomass is fed into a screw zone and transported to the opposite part of the screw. Biomass is mechanically reduced in size by shearing and tearing (Liu et al., 2013) in the gap between rotor and stator, or finally in the extrusion head. Friction forces rise in temperature; as a result, moisture content decreases and generates particle agglomeration, thus increasing mean particle size (Gu et al., 2019). Figure 7 shows an example of an extruder (Kratky, 2020).
Along the length of the screw, there can be openings so chemicals or any other treatment can be implemented and increase the efficiency of the pretreatment (Zheng and Rehmann, 2014). For example, Ä mmä lä . Other authors used this technology to pretreat corn stalks to isolate xylan. It was found that using extrusion led to a higher xylan extraction than grinding but slightly lower than disc refining; the same trend was found for the CrI . Table 2 shows the references consulted regarding the screw extruder.

Knife mill
A knife mill is a widely used size reduction machine to comminute dry biomass. Biomass is continuously fed into size reduction zones formed by static and rotor knife pairs. As material falls between the static and the rotating blade, it is cut and sheared. Comminuted biomass particles fall through a screen sieve to a balance storage tank. The driest and the most brittle the biomass, the highest is the dominance of the cutting principle. As biomass moisture increases, it becomes more elastic. Therefore, cutting effect dominance is reduced, and shearing becomes the dominant size reduction principle.
Additionally, moisture makes biomass sticky. As a result, the size reduction efficiency is reduced owing to its effect associated with clogging screen sieve. Nevertheless, this kind of mill has been used by many authors as it can be operated at a high production rate and is easily performed. Figure 8 shows the milling chamber of a knife mill (Kratky, 2020).    Garuti et al. (2022) used knife milling to pretreat mixed seeds for methane production. They found out that 99% of the particles bigger than 5 mm were reduced after milling. Additionally, a 13% on methane yield was obtained (Garuti et al., 2022). Chuetor et al. (2021) used size reduction to treat rice straw with NaOH and increase cellulose concentration from 34.57% to 66.83% (mg/g Dried Mass) (Chuetor et al., 2021). Knife milling allowed the rice straw to be processable and increased specific surface area. Bianchini et al. (2021) used knife milling as a pretreatment so feedstock could be further valorized into CH 4 . The authors used additional size reduction equipment to obtain the most suitable size to produce biogas in this article. It was found that fines (<300 mm) had the highest CH 4 yield and purity (Bianchini et al., 2021). The typical final particle size after knife milling depends on the sieve installed, but from the references found, the size can be reduced to 100 mm (Garuti et al., 2022). Table 3 shows the articles consulted regarding the knife mill.

Hammer mill
Biomass enters tangentially to rotor hammers to receive a glancing impulse, dynamic effect of pressure force, to send it spinning toward a breaker plate, at which it is broke. Therefore, the primary mechanism responsible for the size reduction is breaking. As a result, comminuted biomass continuously and fractured pieces pass through a sieve. Regarding moisture, literature consulted showed that it has a negative effect on hammer milling, like knife mills. Furthermore, it is more harmful to hammer mills because of the size reduction mechanisms. Moisture makes biomass sticky, and it might adhere to the walls. As a result, these mills are more suitable for brittle material. Figure 9 shows the milling chamber of a hammer mill (Kratky, 2020). Luo et al. (2021) used this technology to observe the influence of particle size on methane production. It was found that the sieve of 3 mm, where most of the particles ranged from 0.6 to 0.25 mm, was the most suitable

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size to perform the anaerobic digestion. It was not the highest methane yield (176.47 vs. 166.07 mLCH 4 /gVS for 1 and 3mm mesh, respectively). However, regarding energy consumption, it was the most efficient one. Size reduction also reduced the crystallinity of the biomass from 24.65 to 15.31% for the smallest particle size (Luo et al., 2021). Al Afif and Pfeifer, 2021 used hammer-milled cotton stalks to produce methane. It was found that the process increased the yield of biogas from 211 to 236 NL/Kg$VS and methane yield from 113.9 to 127.4 NL/kg$VS (Al Afif and Pfeifer, 2021). Particle size depends on the installed sieve, similar to knife mills. After comminution, the minimum particle found was 0.6 mm (Victorin et al., 2020). However, the most common final particle size ranged between 500 and 250 mm (Luo et al., 2021;Maitra and Singh, 2021). Table 4 shows the references consulted regarding hammer milling.

Roll mill
Roll milling is used mainly for flour production, but it can also be used as pretreatment for the enzyme treatment of biomass. Roll milling consists of a series of opposed cylinders that can have a smooth or a dented surface. Biomass is comminuted continuously and steadily introduced between rolls. Thus, compression and tearing are the primary mechanisms of biomass particle size reduction. Roll mill is applicable only for the comminution of brittle or fibrous biomass. Wet biomass is sticky and usually adheres to the surface of rolls. These rolls are continuously spinning in different directions, and biomass is crushed between the gap of opposed cylinders (Cappelli et al., 2020). Finally, a unique roll milling equipment has been used by other researchers: Szego mill. It is similar to roll milling because it has a moving and a static part (Chen et al., 2016). The moving part is shaped like a screw, and the fixed part is the case. Figure 10 shows a scheme of a Szego mill (Chen et al., 2013).
Inside the case are more than one screw-like cylinders that rotate around its axis and the axis of the cage. Raud et al. (2020) used this technology to pretreat barley straw with three different approaches: dry, wet, and liquid nitrogen assisted. Dry milling obtained the highest BMP (from 269 to 292 L CH 4 /kg raw material). However, the highest production rate was obtained with liquid nitrogen assisted (Raud et al., 2020). The lowest particle size obtained from the bibliography was 17.25 mm (Bai et al., 2020).
Regarding roll milling, Bojani c et al. (2021) studied and optimized the roll milling process for wheat flour production regarding yield and energy consumption (Bojani c et al., 2021). However, this technology has been used for wheat production and to increase biorefinery options for biomass. For example, Victorin et al. (2020) used roll milling to reduce the particle size of wheat straw, and as a result, it increased the biogas potential of the biomass. As a result, the generated biochemical methane potential rose from 237 to 287 NmL CH 4 /g VS, the highest among the size reduction treatments studied by the authors (Victorin et al., 2020). Furthermore, other authors have used a particular kind of roll milling by changing the surface of one of the cylinders (Tsapekos et al., 2018) to use grass for biogas production, increasing CH 4 yield from 305 mL/gVS to 367 mL/gVS and from 297 to 376 mL/gVS for both configurations. Table 5 shows references consulted on the roll mill. This type of mill uses high rotational speed to reduce the size of the material. The material enters the mill from the upper part. Inside the milling chamber, there is a rotor with blades that spins at high speed, thus the size reduction mechanisms are cutting and breaking. When the biomass enters the chamber, the mill's blades cut the material, and the resulting particles are thrown at the mill's walls. The mill wall has a mesh that keeps the particles inside until their size is small (Fernando and Manthey, 2022). Figure 11 shows a schematic representation of a centrifugal mill. Ivanchenko et al. (2021) used this comminution technology to treat a mixture of vegetable waste with sewage sludge to produce biogas. It was found that fermentation time decreased from 25 to 12 days after comminution, and biogas production increased by 41% (Ivanchenkoet al., 2021). Other authors used this process to increase the solid load of biomass for bioethanol production. Using the smallest size (% 2.5 mm) had a 16.9% yield glucose concentration, higher than when using the largest size. Additionally, it was discovered that increasing solid loading from 10 to 35% led to a 460% increase in glucose concentration (Hoppert and Einfalt, 2021). Table 6 shows the references to the centrifugal mill.

EQUIPMENT USED FOR BATCH MILLING
The mills with freely set elements are batch size reduction machines that can be applied to comminute biomass independently of its moisture. The ball chamber is a crucial part of the mill. It is fed by the proper iScience Review amount of biomass and grinding elements (steel spheres or rolls). The closed chamber with biomass and grinding parts starts to rotate. A cylinder slowly rotates and moves grinding elements, crushing biomass in the fall. Biomass is also comminuted by its friction between grinding elements and between elements and shells. These mills operate in batch mode, meaning that the final product does not continuously come out of the equipment. Figure 12 shows the milling chamber with the free elements and all the forces that intervene in the process (El-Eskandarany, 2015).

Ball mill
The balls and the feedstock are introduced in a cylinder in a large ratio. Then, the cylinder starts moving, rotating around its central axis. The size reduction occurs by the friction forces between the cylinder and the balls and between the balls themselves. The mechanism responsible for the size reduction depends on the speed of the mill. If the speed is low, the grinding material roll over itself. Thus, the primary size reduction mechanism is tearing between the balls, feedstock, and mill wall. As speed increases, it reaches a point where the centrifugal force makes the grinding material reach the highest point inside the cylinder and fall under the influence of gravity. Thus, breaking becomes an additional mechanism responsible for size reduction. Finally, if the speed is high enough, the grinding material does not fall and distributes along the surface of the chamber. Thus, tearing becomes the only size reduction mechanism but is less efficient than low-speed abrasion. This effect is shown in picture 12. Generally, it is assumed that the optimal speed for size reduction is the one that makes the grinding material reach the maximum height inside the grinding chamber, so abrasion and impact are ensured (Lomovskiy et al., 2020).
Regarding the composition of the grinding material, most of the authors use zirconia (Ji et al., 2018a;Wang et al., 2021;Yu et al., 2019); however, steel (Wu et al., 2021), agate (Yang et al., 2022), aluminum oxide (Kobayashi et al., 2021) and others (Rajaonarivony et al., 2021), can be used. Over the last years, ball milling has been extensively used as size reduction pretreatment for biomass processing, not only because it is easy to perform but also because it can be used with chemicals (mechanochemical) to increase the efficiency of posterior treatments. For example, Xiao et al. (2020) used ball milling on bamboo residues to increase enzymatic saccharification, reducing the CrI of biomass from 71.3% to 9.5%   iScience Review observe the structural changes in willow. Additionally, a total reducing sugars yield of 14.8% was obtained compared to the 1.9% yield without mechanochemical treatment (Lempiä inen et al., 2020). Regarding particle size after milling, it was found that this technology can achieve tiny particle size. The lowest was 9.66 mm . However, the most common particle size obtained from ball milling was around 20 mm (Rajaonarivony et al., 2021), even when the initial particle size was very large (Navarro-Mtz et al., 2019).

Rod mill
In this type of mill, the free set elements are rods. As the milling chamber rotates, the rods move, clashing with each other and the material. The main mechanisms responsible for the size reduction are abrasion and impact. Not many references regarding rod milling and biomass processing for enzymatic production have  been found. However, Bai et al. (2020) used this technology as pretreatment for pyrolysis and biochar production. It was found that torrefaction + rod milling leads to the best results in bio-oil characterization and composition and morphological properties of the biochar (Bai et al., 2020). Table 7 shows the references consulted on ball and rod milling. mL/gVS a (untreated 33.9 mL/gV a S) (Tsapekos et al., 2018) Grass clippings ND a Methane yield 326 mL/gVS a (untreated 33.9 mL/gVS a ) (Tsapekos et al., 2018) Wheat straw ND a Methane yield 255 mL/gVS a (untreated 33.9 mL/gVS a ) (Tsapekos et al., 2018) Digested biofibres ND a Methane yield 42 mL/gVS a (untreated 33.9 mL/gVS a ) (Tsapekos et al., 2018) a N.D., Non-Determined; CrI, Crystallinity Index; gVS, grams of Volatile Solids. Figure 11. Example of a centrifugal mill (Nadutyi et al., 2019) (1) rotor, (2) rotating shaft, (3) disintegration chamber, (4) central gap, (5) feed channel, (6) multichannel boot device, and (7) main channels. Under creative commons license.

A CRITICAL OVERVIEW OF SIZE REDUCTION MACHINES
The review summarized information about size reduction machines' applications to reduce biomass particles and the effect of mechanical size reduction on process efficiency of subsequent technological pathways. Ball mill and disc refiners are the most conventional mechanical size reduction machines applied on a laboratory scale, followed by knife and hammer mills. Regarding ball or disc milling, it can be stated that their use allows us to reach biomass particle size in tents and even lower, hundreds of micrometers. Nevertheless, their application potential in industrial biorefineries is minimal.    Ball mill is an advantageous batch size reduction machine allowing biomass comminution at any moisture. However, it shows the highest specific energy demand, around thousands kWh/t of biomass. In addition, its productivity is limited by the residence time of a given batch, typically in tenths of minutes (Kratky and Jirout, 2011).
Disc refiner is the least reliable as biomass usually clogs the gap. When blocked, temperature increases because of heat dissipation, thus, potentially damaging biomass, especially wet and fibrous biomass. Therefore, it is best suitable for dry biomass. Specific energy demand usually meets the values of hundreds of kWh/t for straw-based biomass (Kratky and Jirout, 2011).
The balance between suitable particle size and subsequent process efficiency was studied in several reports. Biomass particle size between 0.03 and 10 mm is essential for fermentation (Oyedeji et al., 2020). Miao et al. (2011) present the need for 0.5-3.0 mm in corn stover for bioethanol production technology. Sharma et al. (1988) reported biomethane yields for 362 Nm3 t-1 T.S. for particles of 0.088 mm, 360 Nm3 t-1 T.S. for particles 0.40 mm, 350 Nm3 t-1 T.S. for particles 1 mm, 330 Nm3 t-1 T.S. for particles 6 mm a 235 Nm3 t-1 T.S. for particles of 30 mm. Regarding these results, particle size under 1 mm can be disadvantageous in fermentation process control. Izumi et al. (2010) found that when particle size is smaller than 1 mm, the hydrolytic microorganisms are intensively affected by the smallest particles. Lower fatty acids are formed rapidly during their degradation, the pH of the substrate drops sharply, and the iScience Review methanogenesis process is inhibited. Regarding laboratory results, the biomass particles of units in mm seem to be a suitable particle size for biomass treated in industrial lignocellulosic biorefineries. Knife or hammer mills are, therefore, suitable mechanical size reduction machines. These machines ensure continual processing of biomass with moisture up to 25% w/w in high volumes under the least specific energy demands (Kratky and Jirout, 2011) being in units of tenths of kWh/t for straw-or wood-based biomass.

BIOMASS FINAL TREATMENTS
The treatments mentioned above are usually coupled with the final treatment. From the literature revised by the authors, most of these end-user processes focus on biofuels and biogas. Thus, fermentative and enzymatic processes are the most common end-use processes. Figure 13 shows the end-use processes after mechanical pretreatment.
As shown in Figure 13, enzymatic and fermentative processes accounted for 47% and 15% of the revised articles, respectively. However, there are also other options so that biomass could be valorized. For example, the chemical path (23% of the articles consulted chose this path) aims to obtain fine chemicals from the hemicellulose and cellulose sugars or aromatic compounds from lignin. Shen et al. (2020) used mechanochemical treatments to increase the production of 5-Hydroxymethylfurfural from cellulose (Shen et al., 2020). Other authors implemented mechanical treatments to increase the amount of silica recovered from LCB ashes (Park et al., 2021). Thermal treatments represented 9% of the references consulted, including technologies such as pyrolysis or microwave (Mayer-Laigle et al., 2020;Yang et al., 2022). Finally, other treatments represented 6% of the references consulted and focused on the obtention of micro and nano cellulose (Ä mmä lä et al., 2019;Ferreira et al., 2020). However, enzymatic and fermentative are the most used among the references studied. This review focuses on these valorization paths. Figure 14 shows how mechanical treatments improve the effectiveness of enzymes.
As can be seen from the previous illustration, mechanical treatments break down the cell wall to expose fibers from the lignin-cellulose complex making it more accessible for microorganisms or enzymes.

Enzymatic treatment
Enzymes act as catalyzers, increasing the rate of biological reactions by decreasing the activation energy under mild conditions. Depending on the reaction they catalyze, there are several enzymes: Oxidoreductase, transferases, hydrolases, lyases, ligases, and isomerases (Blanco and Blanco, 2017).
When used for the treatment of LCB, enzymes usually focus on the cleavage links of cellulose/hemicellulose, resulting in simpler molecules, so the efficiency of the following process is increased. However, using enzymes on raw LCB usually gives poor performance (Maitra and Singh, 2021) because of the recalcitrant characteristics of this material. Therefore, LCB needs treatment to increase the performance of enzymes. Mechanical treatments do not inhibit the activity of enzymes as they disrupt the molecules mechanically Figure 13. End-use treatments for biomass after mechanical treatments, found in bibliography from most common to least common Enzymatic (47%), chemical (23%), fermentative (15%), thermal (9%), and other treatments (6%).

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iScience 25, 104610, July 15, 2022 iScience Review and do not change the molecules chemically. Furthermore, mechanical treatments have the highest mass yield of all the pretreatments. Sitotaw et al. (2022) studied the possibility of using mechanochemical treatments to improve the performance of enzymatic treatments. It was discovered that mechanical treatments improve cellulose release compared with untreated material. Furthermore, the mechanochemical approach improved sugar yield by up to 86%, which was quicker (Sitotaw et al., 2022). Other researchers used mechanical treatments to increase corn stover saccharification by thread rolling. It was found that this process can increase enzymatic activity. Bigger particles had lower efficiency regarding saccharification (17.5% sugar release for 20-40 mesh) than smaller particles (50.69% sugar release for 60-80 mesh) (Deng and Li, 2021). However, enzymatic hydrolysis is not only used for saccharification or glucose release. This method can also be applied to dissolving pulp to increase the accessibility of cellulose. As dissolving pulp is high-purity cellulose, it also has high crystallinity, which hinders the action of chemicals from modifying this structure. According to Wang et al.(2020), mechanical treatments coupled with enzymes can reduce crystallinity from 68.8 to 47.1% leading to an increase in Fock's reactivity (which is related to the consumption of CS 2 during viscose production) from 54.8 to 78% . However, performing an enzymatic treatment on LCB is not enough for the residues to be valorized. Therefore, there must be a definitive treatment for the LCB to be valorized, and this treatment is usually the fermentation of the released sugars.

Fermentative treatments
Fermentative treatments use microorganisms (bacteria or fungi) to valorize LCB through anaerobic digestion (A.D.). These microorganisms use carbohydrates from LCB as substrate, and, as a result, products are obtained. The main advantages of this process are the low energy consumption and the low waste generation (Llano et al., 2021). However, it needs large equipment and high residence times to complete the reaction (Amin et al., 2017). Again, owing to the recalcitrant nature of LCB, A.D. is usually performed with previous treatments, so reducing sugars can become more accessible (Luo et al., 2021). Authors have used mechanical treatments before A.D., i.e., Garuti et al. (2022) analyzed several size reductions equipment to calculate the efficiency of the process regarding energy. After mechanical treatments, it was found that methane yield increased from 1% to 13%, and the maximum methane production increased from 4% to 48%. Additionally, every equipment used for size reduction led to a positive energy balance (Garuti et al., 2022). Generally, A.D. aims to obtain methane or biogas. However, other authors used A.D. to produce hydrogen by photofermentative bacteria. Corn stover was used as the substrate, and it was pretreated with ultrafine grinding. It was found that increasing grinding time improved H 2 yield and reduced residence time from 36 to 24 h (Tahir et al., 2021). Navarro-Mtz et al., 2019 used soybean meal, a co-product after extracting oil, after mechanical treatment as culture media for microorganism culture (Bacillus thuringiensis). Mechanical treatment led to an increase of released sugar by 34.1 and 2.5 times more fermentable sugars when compared to untreated texture soybean and commercial soybean meal, respectively. Cell growth also was higher than standard culture media without the generation of inhibitors (Navarro-Mtz et al., 2019).

CONCLUSIONS
This review showed recent research on mechanical treatments, focusing on posterior enzymatic/fermentative treatments. Mechanical treatments effectively release cellulose chains within the LCB structure by physically exposing the fibers without altering their chemical properties. In the literature, enzymatic and fermentative treatments have been extensively used as a final treatment after mechanical treatments. However, it must be noted that these treatments need to be controlled in terms of energy to be economically and environmentally feasible. To overcome this significant drawback, optimizing the energetic requirements to a specific particle size should be performed. This optimization should be performed for each feedstock and mill as biomass feedstock is very heterogeneous in chemical and mechanical properties.
Additionally, selecting the proper size reduction mechanism depending on the feedstock is critical as it can lead to excessive energy use. Ball mill is, by far, the most used size reduction operation when working with LCB because it can lead to smaller sizes and is easy to operate. However, as it is a batch operation, scaling it up to an industrial scale needs extra work than continuous comminution processes. In this sense, knife or hammer mills are more suitable for scaling up to an industrial scale. Target particle size is 1 mm, and energy requirements are generally lower when compared to ball milling. Fermentative and enzymatic treatments are also eco-friendly as no waste is generated. Furthermore, if used to produce biogas or hydrogen, it would help to ameliorate greenhouse gas emissions.